Insect-repelling, bacteriostatic and acarid-removing liposome preparation, and preparation method and application thereof

Abstract

The present application relates to a kind of repellent, bacteriostatic, acaricidal liposome preparation and its preparation method and application, belong to daily chemical technology field.The repellent, bacteriostatic, acaricidal liposome preparation provided by the present application includes liposome content, liposome wall material, liposome curing agent and water, and includes by mass percentage: bactericide 0.1-10%, insecticide 0.1-10%, bacteriostatic agent 1-40%, efflux pump inhibitor 0.1-10%, liposome wall material 0.1-10%, liposome curing agent 0.01-1% and the rest water.The dosage form of the present application is liposome, particle size is small, has very strong penetration performance, can help active ingredient better effect;Its composition component is all low toxicity or natural source and the like higher safety component;And have sustained-release effect, prevent active ingredient burst release, provide safer, more long-acting application performance.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of daily chemicals, in particular to a mite-killing, bacterium-inhibiting and acarus-removing liposome preparation as well as a preparation method and application thereof. BACKGROUND

[0002] Mites are closely related to human health. It has been found that mites can invade the skin and cause rhinophyma or demodicidosis, allergy, urinary mite disease, pulmonary mite disease, intestinal mite disease and scabies, which seriously endanger human health. Therefore, household acarus-removing products are increasingly concerned and used by consumers.

[0003] At present, among a variety of acarus-removing products, 80%-90% are daily chemical products, including acarus-removing sprays, acarus-removing sachets and acarus-removing laundry liquids; in the field of textile printing and dyeing, textile auxiliaries with acarus-removing effects are added to make fabrics have acarus-removing functions and be applied to products such as clothes, bed sheets and sofa covers.

[0004] However, in general, acarus-removing products are either added with chemical acarus-removing agents, which have a strong smell and certain harm to human body and have no long-lasting effectiveness, or are added with plant-derived acarus-removing agents, which have less harm to human body but often have general effects. How to balance safety and effectiveness of acarus-removing products is an urgent industry problem to be solved and is also a core concern of consumers. Commercial repellent products such as insecticides, mosquito repellent and bacterium-inhibiting agents also have the above problems.

[0005] Therefore, it is of high application value in the field of daily chemicals to develop a preparation with good mite-killing, bacterium-inhibiting and acarus-removing effects and less harm to skin. SUMMARY

[0006] The present application aims at overcoming the deficiencies of the prior art and provides a mite-killing, bacterium-inhibiting and acarus-removing liposome preparation as well as a preparation method and application thereof. The mite-killing, bacterium-inhibiting and acarus-removing liposome preparation has strong permeability, can realize low-dose high-effect, has high safety and has a sustained-release effect to provide long-acting application performance, and has high application value in the field of daily chemicals.

[0007] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0008] In a first aspect, the present application provides a mite-killing, bacterium-inhibiting and acarus-removing liposome preparation, which comprises a liposome content, a liposome wall material, a liposome curing agent and water, wherein the liposome content comprises a bactericide, an insecticide, a bacterium-inhibiting agent and an efflux pump inhibitor; the bacterium-inhibiting agent is a polyhydric alcohol; and the liposome curing agent is a C16-C22 fatty acid.

[0009] The anti-insect, anti-bacterial and anti-mite liposome preparation comprises the following components in percentage by mass: bactericide 0.1-10%, insecticide 0.1-10%, bacteriostatic agent 1-40%, efflux pump inhibitor 0.1-10%, liposome wall material 0.1-10%, liposome solidifying agent 0.01-1%, and the balance of water.

[0010] The present application provides a complex functional agent with anti-insect, anti-bacterial and anti-mite effects, which is in the form of liposome; the content of the liposome comprises bactericide, insecticide, bacteriostatic agent and efflux pump inhibitor, wherein the fat-soluble components can be contained between the wall material bilayer, and the water-soluble components can be contained inside the bilayer of the liposome; and a liposome solidifying agent is further added to shape and form a more stable preparation.

[0011] The bactericide provides broad-spectrum bactericidal effect; the insecticide has the effects of anti-insect, insecticide and anti-mite, and has high safety; the polyhydric alcohol bacteriostatic agent not only provides broad-spectrum bacteriostatic effect and synergistic antibacterial effect, but also can prevent the formation of biofilm by biological macromolecules outside the bacteria, thereby reducing the drug resistance of the bacteria, and further achieving the effect of synergistic antibacterial effect; and can also be used as a solvent for dissolving the liposome wall material. The efflux pump inhibitor can interfere with the assembly of the efflux pump on the bacterial cell membrane, block the energy source of the efflux pump, and hinder the substrate from directly passing through the efflux channel, so as to directly interfere with the efflux function of the bacteria, inhibit the activity of the bacteria, and achieve the effect of synergistic antibacterial, bacteriostatic and insecticidal effect.

[0012] The liposome wall material can wrap the above-mentioned bactericide, insecticide, bacteriostatic agent and efflux pump inhibitor, and construct the structure basis of the drug-carrying liposome of the complex agent of the present application. Gram-negative bacteria contain many bacterial outer membranes that are difficult for drugs to penetrate, which is the main reason why most antibacterial drugs are ineffective against gram-negative bacteria. The bacterial cell membrane is mainly composed of lipids (mainly phospholipids and cholesterol), and when the liposome wall material in the present application contacts the bacteria, it can inactivate the permeability barrier of the bacterial outer membrane, so that the liposome wall material can easily carry drugs into the bacteria, thereby reducing the drug resistance of the bacteria and enabling the components to directly play a role in the bacteria, thereby achieving the effect of synergistic effect.

[0013] In addition, the liposome solidifying agent introduced in the present application can adjust the appearance state of the liposome wall material and improve the sustained-release effect of the liposome complex agent of the present application. The liposome solidifying agent used in the present application is a fatty acid substance that can solidify liquid fat. The fatty acid chain length is relatively short, with a chain length of C16-C22, and the interaction force between them is relatively weak. When the temperature decreases, the interaction force between molecules gradually increases, causing the fatty molecules to begin to gather together to form small crystal nuclei. These crystal nuclei continue to grow and eventually form a continuous solid structure connected to each other, causing the liquid fat to change into solid fat. Water acts as a solvent and disperses the components.

[0014] The liposome wrapping and solidification strategy adopted by the present application can significantly improve the insect repellent, bacteriostatic and acaricidal effects of the complexing agent. If the liposome dosage form is not adopted or the liposome solidification is not performed, the overall performance will be significantly reduced. The ratio of the liposome wall material and the liposome solidification agent also has a significant impact on the insect repellent, bacteriostatic and acaricidal effects of the present application. Only a specific ratio can comprehensively achieve better insect repellent, bacteriostatic and acaricidal effects.

[0015] The insect repellent, bacteriostatic and acaricidal liposome preparation provided by the present application can form a drug-carrying liposome structure with small particle size and strong penetration performance, which can help the active ingredients to better take effect and achieve the purpose of low dose and high effect. The insect repellent, bacteriostatic and acaricidal liposome preparation provided by the present application contains antibacterial and bacteriostatic insecticidal synergistic components, which can reduce the drug resistance of the receptor. At the same time, the present application has a sustained-release effect, which can prevent the burst release of active ingredients and provide safer and longer-acting application performance.

[0016] Preferably, the insect repellent, bacteriostatic and acaricidal liposome preparation includes the following components in mass percentage: bactericide 0.5-10%, insecticide 1-8%, bacteriostatic agent 20-35%, efflux pump inhibitor 1-5%, liposome wall material 2-3%, liposome solidification agent 0.2-0.5% and the balance of water.

[0017] As a preferred solution, the insect repellent, bacteriostatic and acaricidal liposome preparation includes the following components in mass percentage: bactericide 5-10%, insecticide 5-8%, bacteriostatic agent 30-35%, efflux pump inhibitor 3-5%, liposome wall material 3%, liposome solidification agent 0.3-0.5% and the balance of water.

[0018] Preferably, the bactericide is at least one of dichlorisobutyl, phenoxyethanol, polyhexamethylene hydrochloride, sodium benzoate, potassium sorbate, hydroxybenzoic acid, hydroxybenzyl propyl, methyl isothiazolinone, methyl chloroisothiazolinone, DMDM hydantoin, chlorphenesin.

[0019] Preferably, the insecticide is at least one of natural menthol, pyrethrin, pepper extract, onion extract, orange peel extract, garlic extract, honeysuckle extract, sophora extract, ginkgo extract, rotenone extract, wild wormwood extract, castor seed extract, datura extract.

[0020] Preferably, the bacteriostatic agent is at least one of glycerol, ethanol, propylene glycol, butanediol, pentanediol, hexanediol.

[0021] The bactericide, bacteriostatic agent and insecticide provided by the present application are all low-toxicity or natural-source ingredients, which are safer, less harmful to the skin and non-irritating to the skin, and are more suitable for the daily chemical field.

[0022] Preferably, the efflux pump inhibitor is at least one of glycyrrhizin, methylpyrrolidine, artesunate, antisense nucleic acid, oligonucleotide, amikacin.

[0023] Preferably, the liposome wall material is at least one of lecithin, hydrogenated lecithin, hydroxylated lecithin, hydrogenated lysolecithin, lysolecithin, soybean lecithin.

[0024] Preferably, the liposome solidifying agent is at least one of palmitic acid, linoleic acid, linolenic acid, oleic acid, stearic acid, arachidic acid, behenic acid.

[0025] In a second aspect, the present application provides a preparation method of the above-mentioned insect-repelling, bacteriostatic and acarid-removing liposome preparation, comprising the following steps:

[0026] (1) dispersing the bactericide, the insecticide and the efflux pump inhibitor in deionized water, heating and keeping warm, and mixing uniformly to obtain phase A;

[0027] (2) dissolving the liposome wall material in the bacteriostatic agent, heating and keeping warm, and mixing uniformly to obtain phase B;

[0028] (3) adding the phase B drop by drop into the phase A under heating and keeping warm and stirring to obtain phase C;

[0029] The temperature of the heating and keeping warm is 35-65℃;

[0030] (4) homogenizing the phase C to obtain phase D, then heating to 45-75℃, dispersing the liposome solidifying agent in the phase D, and cooling to 15-30℃ to obtain the insect-repelling, bacteriostatic and acarid-removing liposome preparation.

[0031] The insect-repelling, bacteriostatic and acarid-removing liposome preparation prepared by the above-mentioned preparation method can be prepared into an insect-repelling, bacteriostatic and acarid-removing liposome preparation with stable dosage form and good sustained-release effect, so that the drug-carrying liposome can stably release the functional components for a long time and achieve longer application performance.

[0032] Preferably, the cooling time is 0.5-3h.

[0033] In a third aspect, the present application provides application of the above-mentioned insect-repelling, bacteriostatic and acarid-removing liposome preparation in the field of daily chemicals.

[0034] The insect-repelling, bacteriostatic and acarid-removing liposome preparation provided by the present application has safe and long-acting comprehensive insect-repelling, bacteriostatic and acarid-removing effects, and can achieve low-dose high-effect, and can be further added and applied in various products in the field of daily chemicals, such as laundry liquid and pet shampoo, and has high application value.

[0035] Compared with the prior art, the present application has the following beneficial effects:

[0036] The present application provides a complex agent with good insect expelling, bacterium inhibiting and acarus removing effects by introducing liposome wall materials and liposome solidifying agents and defining each functional component and the proportion, the components of which are low-toxic or natural sources, high in safety and non-irritating to the skin, have strong penetration performance, contain antibacterial and bacteriostatic and insecticidal synergistic components, can reduce the drug resistance of receptors, comprehensively realize low-dose high effect, have stable and long-acting sustained release effect, can prevent the burst release of active ingredients, provide safe and long-acting application performance, are more suitable for the use requirements of daily chemicals, and have high application value. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 It is a transmission electron microscope graph of the insect expelling, bacterium inhibiting and acarus removing liposome preparation in Example 1.

[0038] Figure 2 It is a particle size distribution graph of the insect expelling, bacterium inhibiting and acarus removing liposome preparation in Example 1.

[0039] Figure 3 It is a minimum inhibitory concentration MIC result graph of the insect expelling, bacterium inhibiting and acarus removing liposome preparation in Examples and comparative examples.

[0040] Figure 4 It is a schematic diagram of the sticky board used in the acarus removing test in the effect example.

[0041] Figure 5 It is an acarus removing rate result graph of the insect expelling, bacterium inhibiting and acarus removing liposome preparation in Examples and comparative examples at different concentration gradients.

[0042] Figure 6 It is a release rate curve graph of the active ingredients of the insect expelling, bacterium inhibiting and acarus removing liposome preparation in Example 1, Comparative Example 1 and Comparative Example 2 in water. DETAILED DESCRIPTION

[0043] In order to better illustrate the purposes, technical solutions and advantages of the present application, the present application will be further described below in combination with specific examples. In the following examples, the test methods used are conventional methods unless otherwise specified; and the materials, reagents and the like used are reagents and materials available through commercial channels unless otherwise specified.

[0044] Example 1

[0045] One embodiment of the insect expelling, bacterium inhibiting and acarus removing liposome preparation of the present application, the insect expelling, bacterium inhibiting and acarus removing liposome preparation described in the present embodiment consists of the following components in mass percentage: bactericide 1%, insecticide 1%, efflux pump inhibitor 1%, bacteriostatic agent 20%, liposome wall material 2%, liposome solidifying agent 0.2% and the balance of water.

[0046] The preparation method of the insect expelling, bacterium inhibiting and acarus removing liposome preparation described in the present embodiment is:

[0047] (1) Take 1 g dichlorophen, 1 g natural menthol, 1 g glycyrrhizic acid and disperse them in 74.8 g deionized water, keep the temperature at 45°C, and keep the stirring speed at 500 rpm / min to prepare phase A1;

[0048] (2) Take 2 g of soybean lecithin and uniformly dissolve it in 20 g of propylene glycol, keep the temperature at 45°C, and prepare phase B1;

[0049] (3) Keep the temperature at 45°C, keep the stirring speed at 500 rpm / min, and add phase B1 dropwise into phase A1 to prepare phase C1;

[0050] (4) Homogenize phase C1 by high-pressure homogenizer, the homogenization pressure is 1000 bar, and prepare phase D1;

[0051] (5) Warm phase D1 to 55°C, uniformly disperse 0.2 g of palmitic acid in it, keep the stirring speed at 50 rpm / min, slowly cool to 30°C, and the cooling time is 0.5 h to obtain the insect-repelling, bacteriostatic and acarid-removing liposome preparation, which is called "example 1 sample" hereinafter.

[0052] The transmission electron microscope image of the insect-repelling, bacteriostatic and acarid-removing liposome preparation of example 1 sample is as shown in Figure 1 , and the particle size distribution diagram is as shown in Figure 2 . It can be seen that the insect-repelling, bacteriostatic and acarid-removing liposome preparation is successfully prepared, and the average particle size is 95 nm.

[0053] Example 2

[0054] An embodiment of the insect-repelling, bacteriostatic and acarid-removing liposome preparation of the present application, the insect-repelling, bacteriostatic and acarid-removing liposome preparation of the present embodiment consists of the following components in mass percentage: bactericide 5%, insecticide 5%, efflux pump inhibitor 3%, bacteriostatic agent 30%, liposome wall material 3%, liposome solidifying agent 0.3%, and the balance is water.

[0055] The preparation method of the insect-repelling, bacteriostatic and acarid-removing liposome preparation of the present embodiment is as follows:

[0056] (1) Take 5 g of sodium benzoate, 5 g of natural menthol, 3 g of methyl pyrrolidone and disperse them in 53.7 g of deionized water, keep the temperature at 55°C, and keep the stirring speed at 800 rpm / min to prepare phase A2;

[0057] (2) Take 3 g of soybean lecithin and uniformly dissolve it in 30 g of butanediol, keep the temperature at 45°C, and prepare phase B2;

[0058] (3) Keep the temperature at 55°C, keep the stirring speed at 800 rpm / min, and add phase B1 dropwise into phase A2 to prepare phase C2;

[0059] (4) The C2 phase is homogenized by a high-pressure homogenizer at a homogenization pressure of 2000 bar to obtain a D2 phase;

[0060] (5) The D2 phase is heated to 65℃, 0.3g of linoleic acid is uniformly dispersed therein, and the stirring speed is kept at 70 rpm / min, and then the temperature is slowly reduced to 25℃, and the temperature reduction time is 1.5h, to obtain the insect-repelling, bacteriostatic and acarid-removing liposome preparation, which is referred to as "Example 2".

[0061] Example 3

[0062] In one embodiment of the insect-repelling, bacteriostatic and acarid-removing liposome preparation, the insect-repelling, bacteriostatic and acarid-removing liposome preparation comprises, by mass percentage, the following components: a bactericide 10%, an insecticide 8%, an efflux pump inhibitor 5%, a bacteriostatic agent 30%, a liposome wall material 3%, a liposome solidifying agent 0.5%, and the balance being water.

[0063] The preparation method of the insect-repelling, bacteriostatic and acarid-removing liposome preparation is as follows:

[0064] (1) 10g of potassium sorbate, 8g of pyrethrin and 5g of glycyrrhizic acid are dissolved or dispersed in 43.5g of deionized water, heated at 65℃, and kept at a stirring speed of 1000 rpm / min to obtain an A3 phase;

[0065] (2) 3g of hydrogenated lecithin is uniformly dissolved in 30g of glycerol, and heated at 65℃ to obtain a B3 phase;

[0066] (3) The B1 phase is added dropwise into the A3 phase under the condition of heating at 65℃ and keeping the stirring speed at 1000 rpm / min to obtain a C3 phase;

[0067] (4) The C3 phase is homogenized by a high-pressure homogenizer at a homogenization pressure of 2500 bar to obtain a D3 phase;

[0068] (5) The D3 phase is heated to 55℃, 0.5g of stearic acid is uniformly dispersed therein, and the stirring speed is kept at 50 rpm / min, and then the temperature is slowly reduced to 15℃, and the temperature reduction time is 2h, to obtain the insect-repelling, bacteriostatic and acarid-removing liposome preparation, which is referred to as "Example 3".

[0069] Example 4

[0070] An embodiment of the liposome preparation for expelling insects, inhibiting bacteria and removing mites of the present application differs from the embodiment 1 only in that the amount of glycyrrhizic acid is reduced to 0.2 g, and the amount of deionized water is increased accordingly so that the mass percentages of other components remain unchanged, i.e. 0.2% of the efflux pump inhibitor, to obtain the liposome preparation for expelling insects, inhibiting bacteria and removing mites, hereinafter referred to as "the embodiment 4 sample".

[0071] Embodiment 5

[0072] An embodiment of the liposome preparation for expelling insects, inhibiting bacteria and removing mites of the present application differs from the embodiment 1 only in that the amount of soybean lecithin is reduced to 0.5 g, and the amount of palmitic acid is increased to 0.6 g, and the amount of deionized water is adjusted accordingly so that the mass percentages of other components remain unchanged, i.e. 0.5% of the liposome wall material and 0.6% of the liposome solidifying agent, to obtain the liposome preparation for expelling insects, inhibiting bacteria and removing mites, hereinafter referred to as "the embodiment 5 sample".

[0073] Embodiment 6

[0074] An embodiment of the liposome preparation for expelling insects, inhibiting bacteria and removing mites of the present application differs from the embodiment 1 only in that the amount of palmitic acid is increased to 0.5 g, and the amount of diclosan is reduced to 0.5 g, and the amount of deionized water is adjusted accordingly so that the mass percentages of other components remain unchanged, i.e. 0.5% of the liposome solidifying agent and 0.6% of the bactericide, to obtain the liposome preparation for expelling insects, inhibiting bacteria and removing mites, hereinafter referred to as "the embodiment 6 sample".

[0075] Comparative Example 1

[0076] Comparative Example 1 is prepared based on the embodiment 1 without forming a liposome dosage form and without the solidifying step, and the preparation process comprises the following steps:

[0077] (1) 1 g of diclosan, 1 g of natural menthol and 1 g of glycyrrhizic acid are weighed and dissolved or dispersed in 74.8 g of deionized water, and heated at 45°C with stirring at a speed of 500 rpm / min to obtain a phase A4;

[0078] (2) 2 g of soybean lecithin is uniformly dissolved in 20 g of propylene glycol, and heated at 45°C to obtain a phase B4;

[0079] (3) The phase B4 is added dropwise to the phase A4 under heating at 45°C with stirring at a speed of 500 rpm / min to obtain a phase C4, hereinafter referred to as "the comparative example 1 sample".

[0080] Comparative Example 2

[0081] Comparative Example 2 is prepared based on the embodiment 1 without the solidifying step, and the preparation process comprises the following steps:

[0082] (1) 1 g dichlorisone, 1 g natural menthol, 1 g glycyrrhizic acid were weighed and dissolved or dispersed in 74.8 g deionized water, heated at 45 °C, and kept at a stirring speed of 500 rpm / min to prepare A5 phase;

[0083] (2) 2 g soybean lecithin was uniformly dissolved in 20 g propylene glycol, heated at 45 °C to prepare B5 phase;

[0084] (3) B5 phase was added dropwise into A5 phase at 45 °C, and kept at a stirring speed of 500 rpm / min to prepare C5 phase;

[0085] (4) C5 phase was homogenized by a high-pressure homogenizer at a pressure of 1000 bar to prepare D5 phase, hereinafter referred to as "comparative example 2 sample".

[0086] Comparative Example 3

[0087] Comparative Example 3 was prepared on the basis of Example 1 without forming liposomes, and the preparation process included the following steps:

[0088] (1) 1 g dichlorisone, 1 g natural menthol, 1 g glycyrrhizic acid were weighed and dispersed in 74.8 g deionized water, heated at 45 °C, and kept at a stirring speed of 500 rpm / min to prepare A6 phase;

[0089] (2) 2 g soybean lecithin was uniformly dissolved in 20 g propylene glycol, heated at 45 °C to prepare B6 phase;

[0090] (3) B6 phase was added dropwise into A6 phase at 45 °C, and kept at a stirring speed of 500 rpm / min to prepare C6 phase;

[0091] (4) C6 phase was heated to 55 °C, 0.2 g palmitic acid was uniformly dispersed therein, and kept at a stirring speed of 50 rpm / min, and then slowly cooled to 30 °C for 0.5 h to prepare D6 phase, hereinafter referred to as "comparative example 3 sample".

[0092] Comparative Example 4

[0093] Comparative Example 4 was different from Example 1 only in that the amount of glycyrrhizic acid was reduced to 0.01 g, and the amount of deionized water was adjusted accordingly to keep the mass percentage of other components unchanged, i.e. 0.01% of efflux pump inhibitor, to prepare a sample, hereinafter referred to as "comparative example 4 sample".

[0094] Comparative Example 5

[0095] Comparative Example 5 differs from Example 1 only in that the amount of soybean lecithin is reduced to 0.01 g, the amount of palmitic acid is increased to 1.5 g, and the amount of deionized water is adjusted accordingly so that the mass percentages of the other components remain unchanged, i.e., 0.01% for the liposome wall material and 1.5% for the liposome solidifying agent, to produce a sample, hereinafter referred to as "Comparative Example 5 sample".

[0096] Example 1

[0097] To investigate the bacteriostatic efficacy of the insect-repelling, bacteriostatic, and acaricidal liposome preparation provided by the present application, the insect-repelling, bacteriostatic, and acaricidal liposome preparation of the example and the samples of the comparative examples were subjected to the following minimum inhibitory concentration MIC (%) test:

[0098] The test was performed in accordance with the Ministry of Health 2002 edition "Disinfection Technical Specifications" - 2.1.8.3 Minimum Inhibitory Concentration Test (Agar Dilution Method). The test strains were Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Candida albicans, and Aspergillus niger.

[0099] Culture medium preparation: MH agar was used, and it was prepared in accordance with the product instructions, with a pH of 7.2-7.4. Neisseria gonorrhoeae used GC agar base plus 1% additive; other streptococci used MH agar containing 5% (V / V) sheep blood.

[0100] Medicated agar plate preparation: according to the experimental design, different concentrations of antibacterial drugs that had been diluted by the appropriate factor were added to the MH agar that had been heated and dissolved and equilibrated in a 45-50°C water bath, and thoroughly mixed and poured into sterile plates with a thickness of 3-4 mm. The medicated agar plates were usually prepared at a ratio of 1:9, and the drug concentration range was selected as needed. The prepared medicated agar plates should be placed in a sealed plastic bag and stored in a 2-8°C refrigerator for 5 days.

[0101] Inoculum preparation and inoculation: a bacterial suspension with a concentration equivalent to 0.5 McFarland standard turbidity tube was prepared, and then diluted 1:10 to prepare the bacterial solution with a multi-point inoculator (about 1-2 μl) to inoculate the agar plate surface, with a bacterial count of about 104 CFU per point, forming a bacterial spot with a diameter of 5-8 mm. After inoculation, incubate at 35°C for 16-20 h, and observe the results.

[0102] Result judgment: place the plate on a dark, non-reflective surface to determine the test endpoint, with the minimum drug concentration that inhibits bacterial growth being the MIC. On agar plates containing trimethoprim or sulfonamide, a slight bacterial growth can be observed, and the minimum drug concentration that inhibits more than 80% of the bacterial growth compared to the growth control is taken as the endpoint concentration. If there are two or more colonies growing on agar plates containing drugs at a concentration higher than the endpoint level, or no growth on low-concentration drug agar plates but growth on high-concentration drug agar plates, the culture purity should be checked or the test should be repeated.

[0103] The test results of MIC are shown in Table 1 and Figure 3 The lower the MIC concentration is, the better the bacteriostatic effect is.

[0104] As shown in Table 1, Figure 3 It can be seen that:

[0105] (1) The insect-repellent, bacteriostatic and acarid-removing liposome preparations provided by the embodiments of the present application all have good bacteriostatic effects, and the minimum inhibitory concentration MIC (%) is significantly lower than that of the samples in the comparative examples. Among them, the insect-repellent, bacteriostatic and acarid-removing liposome preparation provided by Example 3 has the lowest MIC and the best effect.

[0106] (2) Comparative Example 1 and Comparative Examples 1-3: It can be seen that the MIC of the sample of Example 1 is significantly lower than that of Comparative Examples 1-3. In Comparative Example 2, no liposome stabilizer is added and no liposome stabilization step is performed, which leads to a significant increase in MIC and a decrease in bacteriostatic effect. In Comparative Example 3, the components are the same but do not form liposomes, which weakens the effect on bacterial cells and significantly reduces the bacteriostatic effect. In Comparative Example 1, no liposomes are formed and no stabilizer is added, which further increases the MIC and reduces the bacteriostatic effect. This verifies that forming a liposome dosage form and further stabilizing the liposomes can significantly improve the bacteriostatic effect and reduce the MIC.

[0107] (3) As can be seen from the comparison between Example 1 and Comparative Example 5, the amount ratio of the liposome wall material and the liposome stabilizer also affects the bacteriostatic effect and the MIC. When too little liposome wall material and too much liposome stabilizer are used in Comparative Example 5, the MIC increases by about 50% and the bacteriostatic effect significantly deteriorates.

[0108] Table 1 MIC test results of the samples of examples and comparative examples

[0109] MIC (%) Staphylococcus aureus Pseudomonas aeruginosa Escherichia coli Candida albicans Aspergillus niger Example 1 2 0.8 0.8 1.2 1.2 Example 2 1 0.4 0.4 0.6 0.6 Example 3 0.4 0.16 0.16 0.24 0.24 Example 4 2.1 0.9 0.9 1.3 1.4 Example 5 2.2 1 1 1.4 1.3 Example 6 1.8 0.7 0.7 1.1 1.1 Comparative Example 1 2.8 1.1 1.15 1.6 1.7 Comparative Example 2 2.6 1 1.1 1.5 1.6 Comparative Example 3 2.7 1 1.1 1.6 1.7 Comparative Example 4 4 1.6 1.7 2.4 2.4 Comparative Example 5 3 1.1 1.2 1.8 1.8

[0110] Example 2

[0111] To explore the acarid-removing effect of the insect-repellent, bacteriostatic and acarid-removing liposome preparation provided by the present application, the insect-repellent, bacteriostatic and acarid-removing liposome preparations of the examples and the samples of the comparative examples are prepared into concentration gradients of 0.1%, 0.5%, 1% and 2% with deionized water as the solvent, and the acarid-removal rates are tested. The acarid-removal test method is as follows:

[0112] Take 7 culture dishes, place one culture dish in the center of the sticky board as the center culture dish, and place the remaining 6 culture dishes around the center culture dish in petal shape, and stick them with transparent tape of the same width at the edges between each culture dish (as a bridge). Then fix the 7 culture dishes on the sticky board, and the arrangement order is as shown in Figure 4 .

[0113] In the 6 peripheral culture dishes, the test samples and the control samples are placed at intervals, the test samples are added with corresponding amount of sample, and the control samples are not added with any substance. The test samples are evenly, flatly and tightly laid on the bottom of the culture dishes, and 0.05 g of mite feed is placed in the center of the test samples and the control samples. Then, (2000±200) living mites (adults or / and nymphs) are placed on the center culture dish.

[0114] The combination of the test mites and the feed placed on the adhesive plate is placed in a constant temperature and humidity incubator, with a temperature of (25±2) °C and a relative humidity of (75±5) %. After 24 h of culture, the living mites in the test sample culture dishes and the control sample culture dishes are observed and counted by using a dissecting microscope or a body microscope.

[0115] The calculation formula of the mite removal rate is as follows:

[0116] Q = (B-T) / B x 100%

[0117] Wherein, Q is the mite removal rate %, B is the average number of living mites in the three control samples, and T is the average number of living mites in the three test samples.

[0118] The calculated value of the mite removal rate is taken as the result, and when the calculated value is negative, it is represented as "0%"; when the calculated value is >99%, it is represented as ">99%". The detection results of the mite removal rates of different samples at different concentrations are shown in Tables 2 and Figure 5 .

[0119] It can be seen from Tables 2 and Figure 5 that:

[0120] (1) The mite removal liposome preparations provided by the embodiments of the present application all have good mite removal effects, and a concentration of 0.5% can achieve a mite removal effect of more than 85% after 24 h of treatment, wherein the mite removal effect of the mite removal liposome preparation provided by Example 3 is the best, and a concentration of 0.1% can achieve a mite removal rate of 99%.

[0121] (2) As can be seen from the comparison of Example 1 and Comparative Examples 1-3, the mite removal rates of the liposome preparations in Example 1 at different concentrations are all higher than those of Comparative Examples 1-3, and the advantage of the mite removal rate is more obvious at a lower concentration. This verifies that the formation of the liposome dosage form and the further solidification of the liposome can significantly improve the mite removal effect and achieve a higher mite removal rate at a lower concentration.

[0122] (3) As can be seen from the comparison of Example 1 and Comparative Examples 4 and 5, the use amount proportions of the efflux pump inhibitor, the liposome wall material and the liposome solidification agent in the present application also have a significant influence on the comprehensive mite removal effect of all components, and when the use amount proportions in Comparative Examples 4 and 5 are outside the range of the present application, the mite removal effect of the mite removal liposome preparation is significantly deteriorated.

[0123] Table 2 Test results of repellency rate of the samples of the examples and the comparative examples

[0124]

[0125]

[0126] Example 3

[0127] To explore the repellent effect of the repellent, bacteriostatic and acaricidal liposome preparation provided by the application, the repellent, bacteriostatic and acaricidal liposome preparation of the examples and the samples of the comparative examples were prepared into samples with a mass concentration of 1%, 3%, 5% and 10% respectively using deionized water as a solvent, and the repellent effect was tested.

[0128] Reference was made to GB / T 13917.9-2009 Indoor Efficacy Test Evaluation of Health Insecticide for Pesticide Registration Part 9: Repellent.

[0129] The specific test method is as follows:

[0130] Firstly, the attack test was performed: 300 test insects were put into the mosquito cage, and the back of the hand of the tester was exposed with a skin area of 40 mm x 40 mm, and the rest was strictly shielded. The hand was stretched into the mosquito cage for 2 min, and the test was closely observed. When the mosquito insects landed and their mouthparts were about to pierce the skin, the hand was shaken to drive them away, and it was recorded as 1 test insect landing. If the number of test insects landing was more than 30, the tester and the test insects were qualified for the attack test, and the repellent test could be performed on the tester and the mosquitoes in the cage.

[0131] After the attack test was qualified, the repellent test was performed: 4 or more testers qualified for the attack test were selected (half male and half female, and they should not drink alcohol, tea or coffee before and during the test, and should not use products containing fragrances), and a skin area of 50 mm x 50 mm was drawn on the back of each hand, wherein one hand was evenly coated with the repellent to be tested at a dose of 1.5 pL / cm (liquid repellent sample), and the exposed skin area of 40 mm x 40 mm was strictly shielded, and the other hand was blank. The repellent was applied for 2 h, and the hand was stretched into the mosquito cage for 2 min, and the landing of mosquitoes was observed. Then the test was performed every 1 h, and as long as one mosquito came to suck blood, the repellent sample was judged to be invalid. The effective protection time (h) of the repellent sample was recorded. The control hand was tested first each time, and the test insects qualified for the attack test could continue the test. If the test insects were not qualified for the attack test, qualified test insects were replaced for the test.

[0132] Test result evaluation:

[0133] The repellent effect of the mosquito repellent product is evaluated according to the effective protection time (h) of the repellent agent on the test personnel. The repellent effect is divided into two levels A and B, and the product that does not reach the B level standard is unqualified. A level: effective protection time ≥ 6.0 h, B level: effective protection time ≥ 4.0 h. The repellent test results are shown in Table 3.

[0134] From Table 3, it can be seen that:

[0135] (1) The insect repellent, bacteriostatic and acaricidal liposome preparation provided by the embodiments has good insect repellent effect, and can achieve at least 4 h of insect repellent protection time at a concentration of 1%; among them, the insect repellent, bacteriostatic and acaricidal liposome preparations provided in Examples 1-3 and 6 have the best effect and can achieve at least 6 h of insect repellent protection time.

[0136] (2) Comparing the insect repellent effect of Comparative Example 1 and Comparative Examples 1-3, it can be seen that the liposome dosage form and liposome solidification treatment of the insect repellent, bacteriostatic and acaricidal liposome preparation provided by the present application can also significantly improve its insect repellent effect. In Comparative Example 1, no liposome is formed and no solidification is performed; in Comparative Example 2, no liposome solidification is performed; and in Comparative Example 3, no liposome is formed, all of which will cause the insect repellent effect to be significantly worse. This may be related to the fact that the liposome can better act on the skin and form a repellent barrier for the complex insect repellent.

[0137] (3) From the comparison of Example 1 and Comparative Examples 4 and 5, it can be seen that when the amount of the efflux pump inhibitor in Comparative Example 4 and the amount of the liposome wall material and the liposome solidification agent in Comparative Example 5 are not appropriate and exceed the limited range of the present application, the insect repellent test will be unqualified and the effective insect repellent effect for a long time cannot be achieved.

[0138] Table 3 Insect repellent test results of the samples of the examples and comparative examples

[0139]

[0140]

[0141] Example 4

[0142] In order to explore the sustained-release effect of the insect repellent, bacteriostatic and acaricidal liposome preparation provided by the present application, the following release rate test was performed on the insect repellent, bacteriostatic and acaricidal liposome preparations of the examples and the samples of the comparative examples:

[0143] 10 g of Example 1 sample, Comparative Example 1 sample and Comparative Example 2 sample were weighed and added to 100 g of physiological saline (0.9% NaCl sterile aqueous solution). The content of the marker glycyrrhizic acid in the physiological saline was detected at 2 h, 4 h, 8 h, 16 h, 24 h, 32 h and 48 h after addition. The ratio of the actual content to the theoretical maximum content was the release rate of the active ingredient of Example 1 sample, Comparative Example 1 sample and Comparative Example 2 sample.

[0144] The release rate results of the active ingredients in the samples of Example 1, the samples of Comparative Example 1, and the samples of Comparative Example 2 are shown in Table 4 and Figure 6

[0145] It can be seen from Table 4 and Figure 6 that the samples of Comparative Example 1, which do not form a liposome dosage form, are all released completely at about 8 h; the samples of Comparative Example 2, which do not undergo a liposome solidification step, have a slower release rate, but also reach a high release rate of 89% after 48 h; and the insect-repelling, bacteriostatic, and acaricidal liposome preparation of Example 1 has a release rate of the active ingredient of less than 50% after 48 h of treatment with physiological saline, and as can be seen from the curve, it can achieve stable and slow release, prevent burst release of the active ingredient, and provide safer and longer-acting application performance; at the same time, this result also indirectly confirms the formation and successful solidification of the liposome dosage form of Example 1 and the stable liposome morphology.

[0146] Table 4 Release test results of Example 1 and Comparative Examples 1 and 2

[0147] Time / Release (%) 2h 4h 8h 16h 24h 32h 48h Example 1 10 15 19 25 31 37 45 Comparative Example 1 75 95 99 99 99 99 99 Comparative Example 2 40 48 57 76 82 85 89

[0148] Effect Example 5

[0149] To explore the safety of the insect-repelling, bacteriostatic, and acaricidal liposome preparation provided by the present application, the insect-repelling, bacteriostatic, and acaricidal liposome preparation prepared in Example was subjected to multiple skin irritation tests, and the test concentration was 5% mass concentration:

[0150] This item was tested according to the Cosmetic Safety Technical Specification, and the testing agency was the Guangzhou Quality Supervision and Testing Research Institute.

[0151] The results of the multiple skin irritation tests of the samples of Example at a concentration of 5% are shown in Table 5. It can be seen from Table 5 that the results of the multiple skin irritation tests are all non-irritating at an addition amount of 5% mass concentration, indicating that the insect-repelling, bacteriostatic, and acaricidal liposome preparation provided by the present application is mild to the skin and has high safety.

[0152] In summary, the insect-repelling, bacteriostatic, and acaricidal liposome preparation provided by the present application can achieve good bacteriostatic, acaricidal, and insect-repelling effects at a low addition concentration, has a slow-release property, can provide safer and longer-acting application performance, and has mild components and no skin irritation at a high concentration, and therefore has high application value in the daily chemical field.

[0153] Table 5 Skin irritation test results of the insect-repelling, bacteriostatic, and acaricidal liposome preparation of the present application

[0154] 5% wt Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Irritant Non-irritant Non-irritant Non-irritant Non-irritant Non-irritant Non-irritant

[0155] Application Example ​

[0156] The repellent, bacteriostatic and acaricidal liposome preparation provided by the present application can be further applied as an additive in laundry liquid and pet shampoo. For example, the specific formula of the repellent, bacteriostatic and acaricidal liposome preparation of Example 1 and Example 2 can refer to Table 6 and Table 7.

[0157] The repellent, bacteriostatic and acaricidal liposome preparation of Example 1 can impart the function of repelling insects, bacteria and mites to laundry liquid, and can be used for daily washing and maintenance of fabrics such as clothes, bed sheets and curtains.

[0158] The repellent, bacteriostatic and acaricidal liposome preparation of Example 2 can impart the function of repelling insects, bacteria and mites to pet shampoo, and can be used for daily cleaning and maintenance of pets with fur such as cats, dogs and hamsters.

[0159] Table 6 Application formula of Example 1 in laundry liquid

[0160]

[0161]

[0162] Table 7 Application formula of Example 2 in pet shampoo

[0163]

[0164]

[0165] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and do not limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A liposome preparation for repelling insects, inhibiting bacteria, and eliminating mites, characterized in that, The preparation is made from liposome content, liposome wall material, liposome solidifying agent and water, wherein the liposome content comprises at least one of bactericide, insecticide, bacteriostatic agent and efflux pump inhibitor; the bacteriostatic agent is at least one of glycerol, ethanol, propylene glycol, butanediol, pentanediol and hexanediol; the liposome solidifying agent is C16-C22 fatty acid; the bactericide is at least one of dichlorophen, phenoxyethanol, polymethylene glycol hydrochloride, sodium benzoate, potassium sorbate, hydroxybenzoic acid ester, hydroxybenzyl propyl ester, methyl isothiazolinone, DMDM hydantoin and chlorphenesin; the insecticide is at least one of natural menthol, pyrethrin, pepper extract, onion extract, orange peel extract, garlic extract, honeysuckle extract, sophora extract, ginkgo extract, rotenone extract and wild wormwood extract; the efflux pump inhibitor is at least one of glycyrrhizic acid and methyl pyrrolidone; the liposome wall material is at least one of lecithin, hydrogenated lecithin, hydroxylated lecithin, hydrogenated lyso-lecithin and lyso-lecithin; and the liposome solidifying agent is at least one of palmitic acid, linoleic acid, linolenic acid, oleic acid, stearic acid, arachidic acid and behenic acid. The preparation method of the insect-repelling, bacteriostatic and acaricidal liposome preparation comprises the following steps: (1) dispersing the bactericide, insecticide and efflux pump inhibitor in deionized water, heating, keeping warm and mixing to obtain phase A; (2) dissolving the liposome wall material in the bacteriostatic agent, heating, keeping warm and mixing to obtain phase B; (3) adding the phase B drop by drop into the phase A under heating, keeping warm and stirring to obtain phase C; the temperature of heating and keeping warm is 35-65℃; (4) homogenizing the phase C to obtain phase D, then increasing the temperature to 45-75℃, dispersing the liposome solidifying agent in the phase D, decreasing the temperature to 15-30℃ to obtain the insect-repelling, bacteriostatic and acaricidal liposome preparation. The insecticide is at least one of natural menthol, pyrethrin, pepper extract, onion extract, orange peel extract, garlic extract, honeysuckle extract, sophora extract, ginkgo extract, rotenone extract and wild wormwood extract.

2. The liposome preparation for repelling insects, inhibiting bacteria, and eliminating mites as described in claim 1, characterized in that, The efflux pump inhibitor is at least one of glycyrrhizic acid and methyl pyrrolidone.

3. The liposome preparation for repelling insects, inhibiting bacteria, and eliminating mites as described in claim 1, characterized in that, The liposome wall material is at least one of lecithin, hydrogenated lecithin, hydroxylated lecithin, hydrogenated lyso-lecithin and lyso-lecithin.

4. The liposome preparation for repelling insects, inhibiting bacteria, and eliminating mites as described in claim 1, characterized in that, The liposome solidifying agent is at least one of palmitic acid, linoleic acid, linolenic acid, oleic acid, stearic acid, arachidic acid and behenic acid.

5. The anti-insect, anti-bacterial, anti-acarid liposome formulation according to claim 1, wherein the liposome is a multilamellar vesicle (MLV) or a small unilamellar vesicle (SUV).

6. The insect-repelling, bacteriostatic and acaricidal liposome preparation according to any one of claims 1-5 for use in the field of daily chemicals. ​

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